Process for devitalizing soft-tissue engineered medical implants, and devitalized soft-tissue medical implants produced

ABSTRACT

The invention provides methodologies and apparatus for producing devitalized soft-tissue implants where the implant retains metabolically non-viable and/or reproductively non-viable cells, and preferably retains large molecular weight cytoplasmic proteins, such implants produced both in small quantities and in commercializable quantities. Such soft-tissue implants include vascular graft substitutes. A devitalized graft is produced by subjecting the tissue sample to an induced pressure mediated flow of an extracting solution, optionally followed by inducing a pressure mediated flow of a salt solution, then washing the tissue to produce the devitalized graft. The devitalized grafts produced are uniform and non-immunogenic. The inventive method allows for the production of multiple devitalized soft tissue implants, where processing time is significantly less than prior art processes and the number of implants produced per day is increased over prior art processes. In clinical use, the devitalized grafts produced exhibit significantly improved in long-term durability and function, and enhanced recellularization post-implantation.

FIELD OF THE INVENTION

The invention is directed toward methodologies and apparatus for use inthe preparation of devitalized soft tissue implants, i.e. essentiallylacking in reproductively viable cells and/or metabolically viablecells, while preferably retaining reproductively non-viable cells and/ormetabolically non-viable cells and/or large molecular weight cytoplasmicproteins including actin. These devitalized, soft-tissue implants, areproduced in small and commercializable quantities and include vascularand musculoskeletal graft substitutes. These implants may be derivedfrom tissue-engineered soft tissues, tissue products derived from animalor human donors that contain cells which contain or are devoid of valvestructures useful in directing the flow of fluids through tubularvascular prostheses, and/or combinations of natural tissue products andtissue-engineered soft-tissue products. The invention includesmethodologies and apparatus for producing uniform, gently processed,devitalized multiple soft tissue implants, where processing time issignificantly reduced and the number of implants produced per day isincreased. The devitalized grafts produced are significantly improved inlong-term durability and function when used in clinical applications.

BACKGROUND OF THE INVENTION

Numerous types of vascular and musculoskeletal grail substitutes havebeen produced in the last four decades. Vascular graft substitutes haveincluded large and small diameter vascular, blood carrying tubularstructures, grafts containing valvular structures (vein substitutes, andheart valve substitutes) and those lacking valvular structures (arterysubstitutes). The materials out of which these vascular grafts have beenconstructed have included man-made polymers, notably Dacron and Teflonin both knitted and woven configurations, and non-man-made polymers,notably tissue engineered blood vessels such as described in U.S. Pat.Nos. 4,539,716, 4,546,500, 4,835,102; blood vessels derived from animalor human donors such as described in U.S. Pat. Nos. 4,776,853,5,558,875, 5,855,617, 5,843,181, and 5,843,180; and connective tissuegrafts as described in U.S. Patent Application No. 2001/0000804 and U.S.Pat. Nos. 6,267,786, 5,865,849, 5,902,338, 5,922,027, 5,944,755,5,984,858, 4,801,299, and 4,776,853.

The prior art processing methods are directed to decellularizing tissuegrafts, i.e. removing all cellular elements, leaving a tissue matrixfree from cellular elements, and are prohibitively time consuming,easily requiring numerous days, for example anywhere from eight totwenty-one days total processing time. Such long processing times resultin proteolytic degradation of the matrix structures of the processedtissues and may result in the accumulation of residuals from the variousprocessing solutions. Over the past few decades numerous efforts havebeen made to manage the large demand for vascular prostheses in thetreatment of vascular dysfunctions/pathologies and musculoskeletalprostheses in the treatment of orthopedic dysfunctions/pathologies.While vascular prostheses are available for clinical use, they have metwith limited success due to cellular and immunological complications,and the inability to remain patent and functional. These problems areespecially pronounced for small diameter prostheses, i.e. less thanabout 6 mm. Efforts have been directed at removing those aspects ofallograft and xenograft vascular prostheses that contribute toimmunological “rejection” and these efforts have focused primarily onthe development of various “decellularization” processes, which requireunduly burdensome incubation times and vast numbers of reagents. Inaddition, the methods used in the prior art dictate the use of largevolumes of processing solutions which do not lend themselves to theproduction of acellular grafts on a commercializable scale.

SUMMARY OF THE INVENTION

The inventive process described herein produces devitalized graftsincluding but not limited to ligaments, tendons, menisci, cartilage,skin, pericardium, dura mater, fascia, small and large intestine,placenta, veins, arteries, and heart valves. The methodology isadvantageous over prior art techniques in that processing times andconditions have been optimized and reduced. In addition, the economicsof production have been dramatically improved, resulting in largenumbers of uniform, non-immunogenic grafts that repopulate withphysiologically competent recipient cells. The grafts' collagenousmatrix is substantially free from damage and retains mid-molecularweight cytoskeletal proteins for the purpose of acting as achemoattractant to the infiltrating cells, such that those cells thatinfiltrate mature into the appropriate cell type. The grafts are alsosubstantially free from contamination including, for example, free frominfectious agents.

The invention embodies the use of one or more non-denaturing agents, forexample, N-lauroyl sarcosinate, to solubilize the cellular components ofthe tissue and leave the matrix such that it is suitable forrecellularization upon implantation. The invention is directed at aprocess for producing devitalized, soft-tissue implants includingvascular grafts, veins, arteries, heart valves, tendons, ligaments,intervertebral discs, menisci and articular cartilage where processingtimes and conditions have been optimized to dramatically improve theeconomics of production as well as to produce a graft with minimumdamage to the matrix structure of the devitalized graft.

The inventive process includes a method for preparing biologicalmaterial(s) for implantation into a mammalian cardiovascular system,musculoskeletal system, or soft tissue system. The process removesreproductively viable cells and/or metabolically viable cells, whilepreferably retaining reproductively non-viable cells and/ormetabolically non-viable cells and/or large molecular weight cytoplasmicproteins including, for example, actin, constituting variability in theinventive process. By varying the extent of the devitalization process,it is possible to differentially control the recellularization of thedifferent tissue types when implanted in different clinicalapplications. For example, cardiovascular tissues that have beendevitalized prior to implantation, need to recellularize and retain avital cell population to rebuild and maintain the matrix structure, butmust resist calcification and fibrosis. Such tissues must alsorecellularize quickly. Musculoskeletal tissues (tendons for example),used in orthopedic and sports medicine applications, do not need torecellularize as quickly as cardiovascular tissues and do not need to beas metabolically active as cells in cardiovascular tissues, but do needto promote mineralization and frequently fibrogenesis. Devitalizedtendons, for example, are frequently used in replacement ofanterior-cruciate ligaments (ACL substitute grafts) and it is importantto minimize recellularization events and maximize osteointegration ofsuch tissues within the femoral and tibial tunnels created to promote astrong attachment of such tissues to the bones being held together bythe ACL substitute grafts. Thus, musculoskeletal tissues such asdevitalized tendons need to retain a different amount and composition ofavital cells than cardiovascular tissues that have been devitalized.Devitalized tendons that retain non-metabolically vital cells will beslow to recellularize and the remnant cell membranes left in the tissuewill tend to serve as nidi for initiating nucleation of mineraldeposition (calcium phosphate salt crystals) in a manner analogous tothe membrane bound matrix vesicles associated with mineralization ofosteoid in the formation of bone. Therefore, tissue intended to beimplanted in cardiovascular applications may be more extensivelydevitalized than tissue that may be utilized in musculoskeletalapplications. To achieve more extensive devitalization, tissues may beexposed to a longer and/or more severe treatment with endonuclease anddetergents, such as Benzonase® and N-lauroyl sarcosinate in onepreferred embodiment; tissues may be processed using ion exchange resinsto remove additional detergent residuals; and/or tissues may be washedmore extensively. According to one aspect of the invention, the tissuesproduced according to these differential methods result in variedpost-treatment assessments. For example, when processing the lessextensively devitalized tissue, visible cells, cell cytoskeletons andmembrane remnants may be viewed in the histology tests, and DNA contentmay be approximately 5%. In comparison, in a process employing moreextensively devitalized tissue post-treatment histology tests mayexhibit no cells and only residual α-actin as cytoskeleton componentsremaining; and the remaining DNA content may be less than 5% or evenless than 0.5% in one aspect. Thus, according to one aspect of theinvention, tissue (such as a musculoskeletal tissue) may be devitalizedsuch that the tissue may have a greater tendency to recalcify and have aslower rate of recellularization after implantation. On the other hand,according to the invention, other tissue, such as cardiovascular tissue,may be more extensively devitalized to permit rapid recellularizationand a substantially lower tendency to calcify.

The process provides for the production of commercializable quantitiesof devitalized soft tissue grafts for implantation into mammaliansystems. The devitalized tissue contains cytoskeletal proteins such asactin, forming a devitalized, non-soluble matrix. The resultant matrixis comprised of collagens, elastins, hyaluronins, and proteoglycans andall or most of the proteins associated with these components. Thedevitalized tissue produced may be implanted into a mammalian system andrecellularized in vivo, or recellularized in vitro and subsequentlyimplanted into a mammalian system.

One embodiment of the process includes the following steps: isolatingfrom a suitable donor a desired tissue sample of the biologicalmaterial; extracting the tissue with mildly alkaline hypotonic bufferedsolution of an endonuclease such as BENZONASE® (a registered product ofMerck KGaA, Darmstadt, Germany) and one or more non-denaturingdetergents, preferably one or more anionic non-denaturing detergents;washing the tissue with sterile water that passes through a bed ofhydrophobic adsorbent resin and an anion exchange resin followed by awater solution, optionally containing one or more decontaminating agentsincluding, for example, chlorine dioxide, glycerol, isopropanol; orINACTINE™ (INACTINE™ is a trademark of VI Technologies, Inc.,Massachusetts); and stored in a sealed container in a storage solutionoptionally including isotonic saline, glycerol and/or one or moredecontaminating agents.

The invention provides a process for preparing a devitalized soft tissuegraft for implantation into a mammalian system and/or commercializablequantities of devitalized, soft tissue grafts, including extracting asoft tissue sample with an extracting solution including one or morenon-denaturing detergents, for example, one or more non-denaturinganionic detergents, and one or more endonucleases, for example, one ormore recombinant endonucleases, to produce extracted tissue; washing theextracted tissue with water that passes through a bed of hydrophobicadsorbent resin and anion exchange resin followed by treating with awater solution of a decontaminating solution including one or moredecontaminating agents to produce the devitalized soft tissue graft; andstoring the devitalized soft tissue graft in a storage solutionoptionally comprising one or more decontaminating agents.

The invention provides a process for devitalizing soft tissue graftsthat alters the matrix structure of the graft and without inhibitingsubsequent recellularization of the soft tissue graft or mechanicalproperties either in vitro, ex vivo, or in vivo.

The invention also provides a process for preparing a devitalized softtissue graft for implantation into a mammalian system, includinginducing a pressure mediated flow of an extracting solution includingone or more non-denaturing anionic detergents and one or morerecombinant endonucleases, through soft tissue, to produce extractedtissue; inducing a pressure mediated flow of water through a bed ofhydrophobic adsorbent resin and anion exchange resin to produceextracted tissue, and inducing a pressure mediated flow of adecontaminating solution including one or more decontaminating agentsthrough the treated tissue to produce the devitalized soft tissue graft;and storing the devitalized soft tissue graft in a storage solutionincluding one or more decontaminating agents. The invention provides aprocess where the extracting solution is recirculated through the softtissue graft. The invention further provides a process where the rinsesolution is recirculated through a bed of hydrophobic adsorbent resinand anion exchange resin. The invention also provides a process wherethe decontaminating solution is recirculated through the soft tissuegraft.

The invention provides a process for preparing a devitalized soft tissuegraft for implantation into a mammalian system, including extracting asoft tissue sample with an extracting solution comprising one or morenon-denaturing anionic detergents and one or more recombinantendonucleases to produce extracted tissue; washing said extracted tissuewith water passing through a bed of hydrophobic adsorbent resin andanion exchange resin to produce extracted tissue and further washingsaid extracted tissue with a decontaminating solution comprising one ormore decontaminating agents to produce said devitalized soft tissuegraft; and storing said devitalized soft tissue graft in a storagesolution; wherein a devitalized soft tissue graft retaining largemolecular weight proteins is produced. The invention also provides adevitalization process which does not employ a denaturing detergent.

The invention provides a process for preparing a devitalized soft tissuegraft for implantation into a mammalian system, including first inducinga pressure mediated flow of an of extracting solution including one ormore non-denaturing anionic detergents and one or more endonucleases,through soft tissue, to produce extracted tissue; inducing a pressuremediated flow of water to pass through a bed of hydrophobic adsorbentresin an anion exchange resin, to produce extracted tissue; inducing apressure mediated flow of decontaminating solution optionally includingone or more decontaminating agents, through said treated tissue, toproduce said devitalized soft tissue graft; and storing said devitalizedsoft tissue graft in a storage solution.

The invention also provides a devitalization process where saidextracting solution, and/or the decontaminating solution, and/or thewashing solution, is recirculated through said soft tissue graft. Theinvention provides a devitalization process where the non-denaturinganionic detergent includes one or more detergents selected from thegroup consisting of N-lauroyl sarcosinate, deoxychloic acid,taurocholic, glycocholic and cholic acids. The invention also provides adevitalization process where the decontaminating solution comprisessterile endotoxin-free, water and/or water solutions of one or moredecontaminating agents, where the decontaminating agents arenon-reactive towards the one or more non-denaturing detergents.

The invention provides a devitalized tissue raft, including a softtissue sample substantially free from reproductively viable and/ormetabolically viable and/or cellular elements produced by the inventiveprocess where recellularization of the devitalized tissue graft in vivoor in vitro, is enhanced by the chemoattractant properties of theresidual mid-molecular weight cytoskeletal proteins. The inventionfurther provides a devitalized tissue graft, including a soft tissuesample substantially free from reproductively viable and/ormetabolically viable and/or cellular elements.

The invention also provides a devitalized soft tissue sample which is aheart valve, and where the devitalized heart valve leaflets maintainnormal coaptation. The invention also provides a devitalized soft tissuesample which is a vascular conduit valved or unvalved graft whichmaintains native function. The invention also provides a devitalizedsoft tissue sample which is a tendon or ligament that maintains nativeelasticity and native function. The invention also provides adevitalized soft tissue sample which is a meniscus or intervertebraldisc that maintains native elasticity and native function.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a view of one embodiment of the processing chambershowing flow mediated processing of long vein segments.

FIG. 2 illustrates a view of an embodiment of the processing chambershowing flow mediated processing of a heart valve through a deformablebag.

FIG. 3 illustrates a view of another embodiment of the flow-throughprocessing chamber including a resin housing device.

FIG. 4 illustrates a view of devitalization chamber and lid.

FIG. 5 illustrates a view of a resin chamber and housing.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions.

The below definitions serve to provide a clear and consistentunderstanding of the specification and claims, including the scope to begiven such terms.

Non-Denaturing Anionic Detergent.

By the term “non-denaturing anionic detergent” is intended for thepurposes of the present invention, any detergent that does not denatureprotein, has a net negative charge and includes, for example, one ormore detergents selected from the group consisting of N-lauroylsarcosinate, deoxychloic acid, taurocholic, glycocholic and cholicacids.

Decontaminating Agent.

By the term “decontaminating agent” is intended for the purposes of thepresent invention, one or more agents which remove or inactivate/destroyany infectious material potentially present in a biological tissuesample, for example, such agents include but are not limited to one ormore of the following: an antibacterial agent; an antiviral agent; anantimycotic agent; an alcohol, for example, methyl, ethyl, propyl,isopropyl, butyl, and/or t-butyl; trisodium phosphate; a preservativesuch as chlorine dioxide, isopropanol, METHYLPARABIN® (Croda, Inc.),INACTINE™; antimicrobials; antifungal agents; sodium hydroxide; hydrogenperoxide; a detergent; and ultrapure water, where the decontaminatingagent or agents do not chemically alter the matrix components of thesoft tissue grafts.

Essentially Free From.

By the term “Essentially Free From” is intended for the purposes of thepresent invention, a soft tissue graft where the material (for example,cellular elements and infectious materials) removed from the soft tissuegraft is not detectable using detection means known in the art at thetime of filing of this application.

Normal Tissue.

By the term “normal tissue” is intended for the purposes of the presentinvention, a particular soft tissue, for example a vein, artery, heartvalve, ligament, tendon, fascia, dura mater, pericardium or skin,present in a living animal, including for example a human, a pig(porcine), a sheep (ovine), and/or a cow (bovine). Tensile properties,as well as other mechanical properties, of a particular devitalized softtissue graft approximate, that is, are not statistically significantlydifferent from, the tensile properties of that tissue in a living animalthat are essential to the function of that tissue.

Devitalized Soft Tissue Graft.

By the term “devitalized tissue graft” it is intended for the purposesof the present invention, soft tissue including, but not limited to,veins, arteries, heart valves, ligaments, tendons, intervertebral disc,menisci, fascia, dura matter, pericardium, and skin, from any mammaliansource, including but not limited to, a human source, porcine source,ovine, and a bovine source, where the devitalized graft produced isallogenic or xenogenic to the mammalian recipient, and where thedevitalized tissue is essentially free from reproductively and/ormetabolically viable cells, for example, a graft devoid ofreproductively viable cells could contain metabolically viable cellsthat are incapable of increasing the numbers of metabolically viablecells through the normal process of meiosis or mitosis; a graft devoidof metabolically viable cells would, for example, be a graft devoid ofcells capable of engaging in those metabolic activities essential to thenormal function of those cells, i.e. the cells would be metabolicallydead, a metabolically dead cell might still be visible in histologysections appearing similar to a metabolically live cell when viewed withthe use of a microscope; cellular remnants, including nucleic acids,small molecular weight proteins, lipids, and polysaccharides, while thedevitalized tissue retains reproductively non-viable cells and/ormetabolically non-viable cells and/or large molecular weight cytoplasmicproteins, such proteins including for example, actin to act as achemoattractant.

Non-Viable Cells:

By the term “non-viable cells” is intended for the purposes of thepresent invention, cells that are metabolically and/or reproductivelynon-viable. A metabolically non-viable cell is a cell incapable ofengaging in those metabolic activities essential to the normal functionof that particular cell, i.e. the cells would be metabolically dead; ametabolically dead cell might still be visible in histology sections. Areproductively non-viable cell is a cell that is incapable of increasingits numbers.

Cellular Elements:

By the term “cellular elements” is intended for the purposes of thepresent invention, those components including but not limited to nucleicacids, small molecular weight proteins, lipids, polysaccharides, andlarge molecular weight cytoplasmic proteins.

Large Molecular Weight Cytoplasmic Proteins:

By the term “large molecular weight cytoplasmic proteins” is intendedfor the purposes of the present invention, cellular elements that areproteins having a high molecular weight that are present in thecytoplasm of cells, such proteins preferably including those having amolecular weight of from about 50 kD to about 2 million kD, and includefor example actin, desmin and vimentin.

USP Grade Sterile Water:

By the term “USP Grade Sterile Water” is intended for the purposes ofthe present invention, water that conforms to standards set forth in theU.S. Pharmacopia for sterility and chemical composition. Using solutionsof high ionic concentration may result in the precipitation ofsolubilized cell remnants that may impart a tendency to the graft to beimmunogenetic and or act as a nidus of calcification.

Storage Solution:

By the term “storage solution” is intended for the purposes of thepresent invention, a solution for storing the devitalized tissue graftand includes, for example, isotonic saline and/or a decontaminatingsolution optionally including one or more decontaminating agents. Suchsolutions include, for example, solutions of chlorine dioxide, alcoholsolutions, isotonic solutions, polyhydroxy compounds such as glycerol,containing one or more decontaminating agents, the decontaminatingagents including, for example, low concentrations of chlorine dioxide,INACTINE™, 70% isopropanol, or peracetic acid. These solutions may alsobe low molecular weight, water replacing agents including, but notlimited to, glycerol as detailed in U.S. Pat. No. 6,569,200, which isincorporated by reference in its entirety.

Allowash® Solution.

By the term “Allowash® solution” is intended those compositionsdisclosed in U.S. Pat. No. 5,556,379 incorporated herein by reference.Examples of suitable Allowash® compositions include a cleaningcomposition containing about 0.06 wt % polyoxyethylene-4-lauryl ether;about 0.02 wt % poly (ethylene glycol)-p-nonyl-phenyl-ether; about 0.02wt % octylphenol-ethylene oxide and endotoxin free deionized/distilledwater.

The invention provides a process for removing viable cells, cellularremnants, nucleic acids, small molecular weight proteins, lipids, andpolysaccharides, while retaining metabolically non-viable and/orreproductively non-viable cells and/or retaining large molecular weightcytoplasmic proteins including for example, actin, and without resultantdamage to the matrix and/or tissue structure. In one preferred aspect,the actin is left to serve as a chemoattractant and thereby attract theappropriate cell type to infiltrate the graft and repopulate it.

Preferably, the tissue thickness does not exceed about 8 mm, morepreferably does not exceed about 6 mm, and most preferably does notexceed about 4 mm, such that the time intervals described herein aresufficient for the process solutions to penetrate the tissue. Processingtimes may be altered to accommodate thicker tissues. A quantity ofendonuclease is used for a given volume of tissue, such that thequantity is sufficient to digest the DNA and RNA within that specifiedvolume of tissue.

The invention recognizes that the mechanical strength of soft tissuegraft biomaterials resides in the matrix structure of the graft. Thematrix structure of these biomaterials includes collagens, elastins,mucopolysaccharides and proteoglycan components. In a preferred aspectof the invention, the devitalization process does not substantiallycompromise the mechanical strength of the graft. Yet, in anotherpreferred aspect of the invention the devitalization process does notcompromise the mechanical strength of the graft.

Although the description of the invention is directed primarily atprocessing vascular graft materials, it should be appreciated that thisinvention is not restricted to processing of vascular graft materialsand may also be directed to processing non-vascular soft tissue grafts.Such tissue grafts include, but are not limited to, tissues such astendons, fascia, ligaments, menisci, pericardium, intestine, skin, dura,and cartilage. Such soft tissue may be processed by one of ordinaryskill in the art to which the present invention pertains by simplemanipulation of the inventive processing times, without undueexperimentation.

Tissue is processed according to the invention by surgically removingnormal healthy tissues, for example, veins, arteries, heart valves,tendons, ligaments, intervertebral discs, menisci, articular cartilage,etc., from animals or humans. The removed tissue is then transported toa processing facility where the tissue is cleaned of extraneous matterand quickly submersed in the first processing (extracting) solutionwhich includes hypotonic buffered solutions containing one or moreendonucleases, for example BENZONASE®, and one or more non-denaturingdetergents including, for example, N-lauroyl sarcosinate. Other suitablenon-denaturing anionic detergents include deoxychloic acid, taurocholic,glycocholic and cholic acids. Procurement and transport of tissue ispreferably carried out sterilely and is held in a sterile container onwet ice in a solution iso-osmolar to the cellular population of thetissue being procured and transported. Furthermore, antibiotics may beadded to the procurement and transport solution as long as tissue andtransport solution samples are acquired for initial bioburdenassessment. The invention includes the use of one or moredecontaminating agents including for example one or more antibiotics,anti-fungal agents or anti-mycotic agents. Other such agents may beadded during processing if so desired to maintain sterility of theprocured tissues.

According to an aspect of the invention, a process for preparingbiological material for implantation into a mammalian cardiovascularsystem, musculoskeletal system, or soft tissue system, or forrecellularization in vitro, is provided and includes removing cells,cellular remnants, nucleic acids, small molecular weight proteins,lipids, and polysaccharides, while retaining large molecular weightcytoplasmic/cytoskeletal components, and forms an extracellular matrixincluding collagens, elastins, proteoglycans, mucopolysaccharides, andlarge molecular weight cytoplasmic/cytoskeletal proteins. The processincludes, isolating from a suitable donor a desired tissue sample of thebiological material; extracting the tissue with mildly alkalinehypotonic buffered solution of one or more endonucleases, for exampleBENZONASE®, and one or more anionic non-denaturing detergents includingfor example. N-lauroyl sarcosinate. Other suitable non-denaturingdetergents include deoxychloic acid, taurocholic, glycocholic and cholicacids. Thereafter, the tissue is washed with water, which is passedthrough a bed of hydrophobic adsorbent resin and anion exchange resin.The tissue subsequently may be exposed to a solution containing one ormore decontaminating agents, such decontaminating agents including, forexample, chlorine dioxide, and alcohol. The decontaminated devitalizedtissue produced is then stored in a storage solution in a sealedcontainer, the storage solution optionally containing one or more waterreplacement agents such as glycerol and/or decontaminating agents, suchsolutions including, for example, isotonic saline; solutions of chlorinedioxide; alcohol solutions; isotonic solution containing one or moredecontaminating agents, the decontaminating agents including, forexample, low concentrations of chlorine dioxide or 70% isopropanol orethanol.

The invention provides for the removal of cellular components withoutresultant damage to the matrix structure in which the cells resided,while ensuring that the repopulation enhancing large molecular weightcytoplasmic proteins, including cytoskeletal proteins including, forexample, actin, are retained to serve as a chemoattractant.

Preferably, the soft tissue sample thickness does not exceed about 4 mmsuch that the time intervals described herein are sufficient for thesolutions to penetrate the tissue. The concentration of endonucleaseutilized is based on calculations designed at achieving a sufficientquantity of endonuclease within a given volume of tissue which issufficient to digest the DNA within that volume of tissue in a specifiedperiod of time and is not arbitrarily chosen based on volume ofprocessing solution. The inventive process maintains the mechanicalstrength of the soft tissue graft biomaterials in part because theprocess does not detrimentally affect the integrity of the collagenmatrix structure of the graft.

The invention provides for the production of soft tissue grafts, whichare readily repopulated by recipient cells, post implantation, orreadily repopulated in vitro. The inventors surprisingly discoveredproducing a devitalized tissue which retains large molecular weightcytoplasmic proteins results in enhanced repopulation of the devitalizedtissue graft after implantation. The inventors have further discoveredthat controlling the cellular remnants and removing processing reagentsand solubilizable cellular remnants enables cells to repopulate thetissue and the cells repopulating the tissue to remain non-apoptoticfacilitating long-term function and synthesis of new matrix. In oneaspect, in vivo or in vitro recellularization of treated tissue iscontrolled by managing the levels of cellular remnants left in thetissues after devitalization. In this context, cellular remnants mayinclude cells that are incapable of dividing, cells that are incapableof maintaining metabolic function, and cells that do not retain anythingexcept the cytoplasmic skeleton. In one embodiment, the inventors havefound that for devitalized tissues that retain reproductively nonviablecells, the cells undergo apoptosis and degrade over time after beingimplanted. Such tissues may promote osteointegration and may be slowerto recellularize compared to tissues devitalized to retain onlycytoskeletal proteins. On the other hand, devitalized tissues thatretain cytoplasmic proteins and a minor amount of cellular remnants willrecellularize more quickly with minimal recalcification. Thus, dependingon the ultimate application of the tissue to be implanted, thedevitalization process may be controlled to promote or retardrecellularization and to promote or retard osteointegration. Therefore,devitalized musculoskeletal tissues (tendons for example) may preferablybe processed to retain reproductively/metabolically non-vital cells andcardiovascular tissues may be processed to retain cytoplasmic proteinswhere the musculoskeletal tissues will be implanted in sites where thecellular remnants will promote osteointegration and retardrecellularization and cytoplasmic cytoskeletal proteins will not promotecartilage or bone formation and will promote recellularization.

Although the description of this invention is directed primarily atprocessing vascular graft materials and tendons, it should beappreciated that this invention may also be directed to processingfascia, ligaments, menisci, intervertebral discs, pericardium, skin,dura, and cartilage by simple manipulation of processing times andparameters, such manipulation may be readily determined and employed byone of ordinary skill in the art, without undue experimentation.

In the inventive process, normal healthy vessels (veins, arteries, heartvalves, tendons, ligaments, fascia, pericardium, intestine, urethra,etc.) are surgically removed from animals or humans, transported to theprocessing facility where they are cleaned of extraneous matter andimmediately submersed in an extracting solution which contains ahypotonic buffered solution containing one or more endonucleasesincluding for example, BENZONASE®, and one or more non-denaturingdetergents including, for example, N-lauroyl sarcosinate. In that mostsuch vessels and tendons are procured at sites distant from theprocessing facility and that such vessels and tendons may ultimatelyeither be cryopreserved, devitalized or cryopreserved and subsequentlydevitalized, procurement and transport will normally be in a sterilecontainer on wet ice in a solution iso-osmolar to the cellularpopulation of the tissue being procured and transported. One or moredecontaminating agents, including, for example, one or more antibiotics,may be optionally employed in any step of the inventive process, tomaintain sterility of the procured tissues.

FIG. 1 illustrates the processing of a long vein grafts (1), the distalend of the vein is cannulated onto the ribbed attachment (2) of theinlet port (3) and a single suture (4) is used to secure the vein. Anadditional suture line (5) is attached to the proximal end of the veinfor later use in maintaining the vein in an extended state in theprocessing vessel (6). The vein (1) is then removed from the extractingsolution and transferred to the processing vessel (6) that has beentemporarily inverted. The second suture line (5) along with the vein (1)is passed through the processing vessel (6) and secured to a point (7)on the outlet port end (8) of the processing vessel (6). Prior toclosing the processing vessel, a portion of the extracting solution isgently added to the processing vessel and the inlet port (3), withattached vein (1), is then secured. The processing vessel (6) is turnedsuch that the inlet port (3) is down and the outlet port (8) is up andthe vessel (6) is attached to its support racking system via clamps (9).Sterile disposable tubing (10) is attached to the inlet port (3) and topump tubing in a peristaltic pump (11). Further, sterile disposabletubing (12) is attached to the inflow side (13) of the peristaltic pump(11) and to the solution reservoir (14) which will contain all remainingextracting solution. Finally, sterile disposable tubing (15) is attachedbetween the top (outlet) port (8) of the processing vessel (6) and thesolution reservoir (14). Sterile, in-line, filters (16) may optionallybe added at suitable positions in the fluid flow to safeguard sterilityduring processing. The extracting solution is pumped into, through andout of the processing vessel (6) such that flow of fluids through theluminal part of the vein tubule passes into the processing vessel (6) toaffect constant solution change in the processing vessel and out throughthe outlet port (8) to a solution reservoir (14). By processing the veinin an inverted state, air which may be trapped in the luminal space ofthe vein will be induced to exit, thereby facilitating equal access ofthe processing solutions to the vein tissue being processed. Processingof the vein tissue with the extracting solution is preferably carriedout at temperatures ranging from about 4° C. to about 42° C., preferablyfrom about 10° C. to about 37° C., and most preferably from about 15° C.to about 25° C., for time periods ranging from about 1 hour to about 36hours (overnight as necessary to accommodate processing scheduling ofprocessing staff), preferably from about 6 hours to about 30 hours, andmore preferably from about 12 hours to about 24 hours. The extractingsolution is preferably pumped at a flow rate of from about 2 mls/min toabout 200 mls/min, more preferably from about 5 mls/min to about 100mls/min and most preferably from about 30 mls/min to about 60 mls/min.One preferred endonuclease (BENZONASE®) is optimally active between pH 6and 10, and from 0° C. to above 42° C. (Merck literature describingproduct) when provided with 1-2 mM Mg⁺². Thereafter, the tissue isprocessed with a decontaminating water solution which passes through abed of hydrophobic adsorbent resin and anion exchange resin optionallycontaining one or more decontaminating agents including, for example,chlorine dioxide. Under the optional processing procedures, onlysufficient solution need be circulated through the processing vessel toaffect one volume change of solution in the processing vessel. Under theprocessing procedures with the water, this solution should be circulatedthrough the tissue at a temperature of from 0° C. to about 42° C.,preferably from about 10° C. to about 37° C., and most preferably fromabout 15° to about 25° C., for a time period of at least 3 hours,preferably from about 1 to about 36 hours, and most preferably fromabout 3 to about 24 hours.

Following processing with the final processing solution, i.e. water ordecontaminating water solution, the vein is removed from the processingvessel and transferred into storage solution, for example, glycerol, 70%isopropanol, or 0.001% to 0.005% chlorine dioxide in sterile ultrapurewater/isotonic saline, and packaged in a volume of storage solutionsufficient to cover the tissue preventing dehydration or stored betweenminus 80° C. and 4° C. This packaged graft may then be terminallysterilized, for example, using gamma irradiation, if so desired. Arterysegments may be similarly processed, taking into consideration thatveins have valves and the direction of flow must mimic the physiologicalflow of blood through the veins and their valves. Veins also generallyhave a smaller internal diameter than arteries, thus dictating slowerflow rates with veins.

FIG. 2 illustrates processing heart valve grafts. The heart valve (1) isplaced into the deformable processing device (6′) such that the valvedend of the conduit is directed towards the inlet port (3) and thenon-valved end of the conduit is directed towards the outlet port (8).Prior to closing the processing vessel (6′), a portion of the extractingsolution is gently added to the processing vessel. The processing vessel(6′) is turned such that the inlet port (3) is down and the outlet port(8) is up to effect removal of air bubbles, and the vessel (6′) isattached to its support racking system via clamps (9). Steriledisposable tubing (10) is attached to the inlet port (3) and to pumptubing in a peristaltic pump (11). Further, sterile disposable tubing(12) is attached to the inflow side (13) of the peristaltic pump (11)and to the solution reservoir (14) which will contain all remainingextracting solution. Finally, sterile disposable tubing (15) is attachedbetween the top (outlet) port (8) of the processing vessel (6′) and thesolution reservoir (14). Sterile, in-line, filters (16) may optionallybe added at suitable positions in the fluid flow to safeguard sterilityduring processing. The extracting solution is pumped into, through andout of the processing vessel (6′) such that the flow of fluids throughthe luminal part of the heart valve (1) passes into the processingvessel (6′) to affect constant solution change in the processing vessel(6′) and out through the outlet port (8) to a solution reservoir (14).By processing the heart valve (1) in this orientation, air which may betrapped in the luminal space of the valve will be induced to exitfacilitating equal access of the processing solutions to the valvetissue being processed. Processing of the heart valve (1) tissue withthe extracting solution is performed at, for example, a temperature offrom about 4° C. to about 42° C., preferably from about 10° C. to about37° C., and most preferably from about 15° C. to about 27° C., for timeperiods ranging from about one hour to about 36 hours (overnight asnecessary to accommodate processing scheduling of processing staff),preferably from about 6 hours to about 30 hours, and more preferablyfrom about 12 hours to about 24 hours. The extracting solution ispreferably pumped at a flow rate of from about 50 mls/min to about 350rills/min, more preferably from about 100 mls/min to about 275 mls/minand most preferably from about 150 mls/min to about 250 mls/min. Theextracting solution may be supplemented with antibiotics such asPolymixin, Vancomycin and or Lincomycin at concentrations familiar tothose skilled in the art. However, Cefoxitin is inhibitory to theactivity of BENZONASE® and is therefore not used to disinfect the tissueduring devitalization. One preferred endonuclease, BENZONASE®, isoptimally active between pH 6 and 10, and from 0° C. to above 42° C.(Merck literature describing product) when provided with 1-2 mM Mg².Following processing with the extracting solution, the extractingsolution is optionally replaced with water which is recirculated througha bed of hydrophobic resin such as XAD-16® from Rohm and Haas and ananionic exchange resin, AMBERLITE 410®, from Rohm and Haas. Thereafter,the tissue is processed with a decontaminating water solution optionallycontaining one or more decontaminating agents including, for example,chlorine dioxide, antibiotics or isopropanol. Under the optionalprocessing procedures, only sufficient solution (including thehypertonic salt solution) need be circulated through the processingvessel to affect one volume change of solution in the processing vessel.Under the processing procedures with water this solution should becirculated through the tissue at a temperature of from 0° C. to about42° C., preferably from about 20° C. to about 37° C., and mostpreferably from about 20° C. to about 27° C., for a time period of atleast three hours, preferably from about one to about twenty-four hours,and most preferably from about three to about six hours.

Following processing with the final processing solution, i.e. water ordecontaminating water solution, sterile isotonic saline is circulatedthrough the tissue such that the available volume of washing solutionapproximates a 1000-fold dilution of previous solutions. In this finalprocessing step, the heart valve is removed from the processing vesseland transferred into storage solution, for example, water solution ofglycerol, 70% isopropanol, or 0.001% to 0.005% chlorine dioxide insterile ultrapure water/isotonic saline, and packaged in a volume ofstorage solution sufficient to cover the tissue to prevent dehydration.This packaged graft may then be terminally sterilized, for example,using gamma irradiation, if so desired.

For all other soft tissue grafts preferably the thickness does notexceed about 8 mm, more preferable does not exceed 5 mm, and mostpreferably the thickness does not exceed about 2-3 mm. If the thicknessof the tissue graft exceeds about 5 mm, incubation and processing timesneed to be suitably extended. Such incubation and processing times maybe readily selected and employed by one of ordinary skill in the art towhich the present invention pertains without undue experimentation basedon the thickness of the tissue being processed, the type of tissue beingprocessed, and the volume of tissue being processed. Prior to closingthe processing vessel, a portion of the extracting solution is gentlyadded to the processing vessel. The vessel is attached to its supportracking system, for example, via clamps. Sterile disposable tubing isattached to the inlet port and to pump tubing in a peristaltic pump.Further, sterile disposable tubing is attached to the inflow side of theperistaltic pump and to the solution reservoir, which will contain allremaining extracting solution. Finally, sterile disposable tubing isattached between the top (outlet) port of the processing vessel and thesolution reservoir. Sterile, in-line, filters may optionally be added atsuitable positions in the fluid flow to safeguard sterility duringprocessing. The extracting solution is pumped into, through and out ofthe processing vessel such that flow of fluids occurs in close proximityto the surfaces of the soft tissue grafts into the processing vessel toaffect constant solution change in the processing vessel and out throughthe outlet port to a solution reservoir. Processing of the soft tissuegraft with the extracting solution is preferably performed at atemperature of from about 4° C. to about 42° C., preferably from about10° C. to about 37° C., and most preferably from about 15° C. to about27° C., for a period of time preferably of from about 1 hour to about 48hours, (overnight as necessary to accommodate processing scheduling ofprocessing staff), preferably from about 8 hours to about 48 hours, andmore preferably from about 12 hours to about 36 hours. The extractingsolution is preferably pumped at a flow rate of from about 10 mls/min toabout 500 mls/min, more preferably from about 50 mls/min to about 350mls/min and most preferably from about 100 mls/min to about 275 mls/min.One preferred endonuclease (BENZONASE®) is optimally active between pH 6and 10, and from 0° C. to above 42° C. (Merck literature describingproduct) when provided with 1-2 mM Mg⁺². Following processing with theextracting solution, the rinse solution is circulated through and/oraround the tissue at a temperature of from about 4° C. to about 42° C.,preferably from about 10° C. to about 37° C., and most preferably fromabout 15° C. to about 27° C., for a time period of at least 12 hours,preferably from about 8 to about 48 hours, and most preferably fromabout 12 hours to 36 hours. Ultrapure sterile water is circulatedthrough a bed of hydrophobic adsorbent resin and anion exchange resinand/or around the tissue and processing vessel. Following the waterwash, the tissue is optionally processed with a decontaminating solutionand a water replacement agent.

Throughout processing for all tissue grafts, the tissue is processed ata flow rate sufficient to affect a volume change in the processingvessel about every 2-5 minutes, suitable flow rates including, forexample, from about 100 mls/min to about 500 mls/min, preferably fromabout 150 mls/min to about 300 mls/min, even more preferably from about200 mls/min to about 250 mls/min and most preferably about 250 mls/min.Following washing with the decontaminating solution, the soft tissuegraft may be removed from the processing vessel and transferred into awater replacement agent such as glycerol as a storage medium.Alternatively, the storage solutions may be pumped into the processingvessel until the decontaminating solution has been adequately exchangedand the whole processing vessel sealed, sterilized, for example, usinggamma-irradiation, and used as the storage container for distribution.Suitable storage solutions are well known to those of ordinary skill inthe art to which the present invention applies, and such solutions maybe readily selected and employed by those of ordinary skill in the artto which the present invention applies without undue experimentation.The storage containers with solution and soft tissue grafts may beterminally sterilized using methods known in the art including, but notlimited to, gamma irradiation at doses up to 2.5 Mrads.

FIG. 3 illustrates a system for processing heart valve grafts or similarnon-vascular tissue grafts such as tendons. A heart valve ornon-vascular tissue graft, such as a tendon, is placed into thedevitalization chamber (A) using a suitable insert shown in FIG. 4 tominimize the volume processing reagents required to devitalizalize thetissue. The lid (B) is screwed down tightly to engage the o-ring therebyeliminating leakage from the chamber (A). The hydrophobic adsorbentresin and anion exchange resin are added to the resin chamber shown inFIG. 5 and placed in the resin housing (C). There is an o-ring at thetop and bottom of the resin chamber to ensure a secure fit between theresin chamber and the resin housing to force the flow of water throughthe resin chamber. Sterile disposable tubing that is medical grade isattached to ports 101, 103, 105, 106, 107, 109 110 and 111 with 3-waystop cocks inserted in-line (112, 113, and 114). The tubing is attachedto the sipper devices (115, 116) such that the return flow enters theside with the shortest spout and the outbound flow is pulled through thelongest spout. The tubing is placed onto the rollers of the peristalticpump (102) and the clamp lowered to hold the tubing in place. Once thewater and devitalization solution (118) (Buffer, N-lauroyl sarcosinate,polymixin B and BENZONASE®) are connected, all connections are checkedto ensure that they are tight. Pump (102) is turned on and itscalibration is checked. The solution is drawn up the long spout ofsipper (115) proceeds through port (101) and tubing through the rollerassembly of the pump (102) into the tissue chamber through port (103)and proceeds through the insert, which diffuses the liquid through thegrafts, then out the top of the chamber and through port (105) andcontinues past stopcocks (113 and 112) then into the sipper (115)through the short spout and port (106). This cycle continues forapproximately 24 hours at 15° C. at 250 mls/minute. After the 24 hourshave passed, the system is reversed to empty the tissue chamber.Stopcocks (112, 113 and 114) are turned to redirect the flow to and fromthe water reservoir (117) and to direct the flow through the resinhousing chamber (C). The pump flow direction is then returned to theinitial direction, the chamber is filled by the water exiting sipper(116) out the long spout and into the tubing through (111), through therollers on pump (102), through the devitalization chamber (A) into thetissue chamber through port (103) and proceeds through the insert, whichdiffuses the liquid through the gratis, then out the top of the chamberand through port (105) and continues past stopcocks (113) which directsthe flow of water into the resin housing chamber (C) through the resinchamber (108) out port (109) through the tubing and into sipper (116)via the short spout (110) and into the water reservoir (117). This cyclecontinues for 24 hours at 15° C. at 250 mls/minute.

When the tissue is to be removed, the pump is reversed to remove thewater from the devitalization chamber and then the tissue is asepticallyremoved and placed into a storage solution containing 0.001% chlorinedioxide and 80% glycerol. Throughout processing for all tissue grafts,the tissue is processed at a flow rate sufficient to affect a volumechange in the processing vessel about every 2-5 minutes, suitable flowrates including for example of from about 100 mls/min to about 500mls/min, preferably from about 150 mls/min to about 300 mls/min, evenmore preferably from about 200 mls/min to about 250 mls/min and mostpreferably about 250 mls/min. Following washing with the decontaminatingsolution, the soft tissue van may be removed from the processing vesseland transferred into a water replacement agent such as glycerol as astorage medium. Alternatively, the storage solutions may be pumped intothe processing vessel until the decontaminating solution has beenadequately exchanged and the whole processing vessel sealed, sterilizedfor example using gamma-irradiation, and used as the storage containerfor distribution. Suitable storage solutions are well known to those ofordinary skill in the art to which the present invention applies, andsuch solutions may be readily selected and employed by those of ordinaryskill in the art to which the present invention applies without undueexperimentation. The storage containers with solution and soft tissuegrafts may be terminally sterilized using methods known in the artincluding, but not limited to, gamma irradiation at doses up to 2.5Mrads.

The following examples illustrate processing of soft tissue graftsaccording to the invention.

Example 1

Saphenous vein tissues (two) from each leg of an acceptable human donorwere carefully dissected under sterile conditions to remove all visiblefat deposits and the side vessels were tied off using non-resorbablesuture materials such that the ties did not occur in close proximity tothe long run of the vessel. Sutures may restrict the devitalizationprocess and the tissues under the sutures were removed followingdevitalization. For long vein grafts (40-60 cm) (FIG. 1), the distalends of the veins were cannulated onto the ribbed attachment of theinlet ports and single sutures used to secure each vein. Additionalsuture lines were attached to the proximal ends of the veins. The veinswere then removed from the dissecting solution (RPMI 1640, papaverine)and transferred to the processing vessel which had been temporarilyinverted. The second suture line along with the vein was passed throughthe processing vessel and secured to a point on the outlet port end ofthe processing vessel. Prior to closing the processing vessel, a portionof the extracting solution was gently added to the processing vessel andthe inlet port, with attached vein, was then secured. The processingvessel was then turned such that the inlet port was down and the outletport was up and the vessel attached to its support racking system viaclamps. Sterile disposable tubing was attached to the inlet port and topump tubing in a peristaltic pump. Further, sterile disposable tubingwas attached to the inflow side of the peristaltic pump and to thesolution reservoir which contained all remaining extracting solution.Total extracting solution volume approximated 250 ml. Finally, steriledisposable tubing was attached between the top (outlet) port of theprocessing vessel and the solution reservoir. Sterile, in-line, filterswere added at suitable positions in the fluid flow to safeguardsterility during processing. The extracting solution was then pumpedinto, through and out of the processing vessel such that flow of fluidsthrough the luminal part of the vein tubule passed into the processingvessel to affect constant solution change in the processing vessel andout through the outlet port to a solution reservoir. By processing thevein in an inverted state, air which had been “trapped” in the luminalspace of the vein was induced to exit, which facilitated equal access ofthe processing solutions to the vein tissue being processed. Processingof the vein tissue with the extracting solution was performed at 15° C.for 24 hours using a flow rate of the extracting solution of 50 mls/min.The extracting solution consisted of 50 mM. Tris-HCL/Tris Base (pH 8.0),2 mM MgCl₂, 16 mM N-lauroyl sarcosinate, and an endonuclease(BENZONASE®) (203 U/ml). Following processing with the extractingsolution, water was passed through the veins and then through a bed ofhydrophobic adsorbent resin (XAD-2) and anion exchange resin (Amberlite910) at 15° C. for 24 hours at 50 mls/min. Following the rinse step awater replacement agent, glycerol was circulated through the veins inconjunction with a decontaminating agent, 0.001% chlorine dioxide, for 3hours at 15° C. at 50 mls/min. Fluorometry using Pico green (MolecularProbes) was used to determine the percent reduction in DNA. The averagepercent reduction was 99.93%+/−0.03%. In addition, H&E staining andMasson's Trichrome stain were employed to look for residual nuclei andresidual cell remnants, respectively. There were no nuclear remnantsseen on the H&E slides and a minimal amount of desirable cytoplasmicproteins. In addition immunohistochemistry was performed to demonstratethe absence of MHCI/II proteins and the presence of alpha smooth muscleactin.

Example 2

Saphenous vein tissues (two) from each leg of an acceptable human donorwere carefully dissected under sterile conditions to remove all visiblefat deposits and side vessels were tied off using nonresorbable suturematerials such that the ties did not occur in close proximity to thelong run of the vessel. Sutures may restrict the devitalization processand the tissues under the sutures were removed following devitalization.For long vein grafts (33 and 28 cm) (FIG. 1), the distal ends of theveins were cannulated onto the ribbed attachment of the inlet ports andsingle sutures used to secure each vein. Additional suture lines wereattached to the proximal ends of the veins. The veins were removed fromthe dissecting solution (RPMI 1640, papaverin) and Polymixin B. Then theveins were cryopreserved according to current guidelines in RMPI 1640,10% fetal calf serum, and 10% DMSO and control rate frozen at 1° C./minand held in nitrogen vapor until devitalization. Prior to devitalizationthe tissue was thawed and diluted using an AlloFlow® chamber (disclosedin U.S. Pat. Nos. 5,879,876 and 6,326,188, which are incorporated byreference in their entireties herein). The veins were then transferredto the processing vessel, which had been temporarily inverted. Thesecond suture line along with the vein was passed through the processingvessel and secured to a point on the outlet port end of the processingvessel. Prior to closing the processing vessel, a portion of theextracting solution was gently added to the processing vessel and theinlet port, with attached vein, was then secured. The processing vesselwas then turned such that the inlet port was down and the outlet portwas up and the vessel attached to its support racking system via clamps.Sterile disposable tubing was attached to the inlet port and to pumptubing in a peristaltic pump. Further, sterile disposable tubing wasattached to the inflow side of the peristaltic pump and to the solutionreservoir, which contained all remaining first extracting solution.Total processing solution volume approximated 250 ml. Finally, steriledisposable tubing was attached between the top (outlet) port of theprocessing vessel and the solution reservoir. Sterile, in-line, filterswere added at suitable positions in the fluid flow to safeguardsterility during processing. The extracting solution was pumped into,through and out of the processing vessel such that flow of fluidsthrough the luminal part of the vein tubule passed into the processingvessel to affect constant solution change in the processing vessel andout through the outlet port to a solution reservoir. By processing thevein in an inverted state, air which had been “trapped” in the luminalspace of the vein was induced to exit facilitating equal access of theprocessing solutions to the vein tissue being processed. Processing ofthe vein tissue with the extracting solution was performed at 15° C. for8 hours using a flow rate of the extracting solution of 50 mls/min. Theextracting solution consisted of 50 mM Tris-HCl/Tris Base (pH 8.3), 2 mMMgCl₂, 32 mM n-lauroyl sarcosinate, and an endonuclease (BENZONASE®)(406 Units/ml). Following processing with the extracting solution, theextracting solution was replaced with sterile water and was circulatedthrough the tissue and a hydrophobic adsorbent resin (XAD-16®) and anionexchange resin (AMBERLITE 410®) at a flow rate of 50 mls/min for 16hours. Following washing in this final processing step, the vein wasremoved from the processing vessel and transferred into storage solutionof 80% glycerol and 0.001% chlorine dioxide in sterile ultrapure waterand packaged in a volume of this solution sufficient to cover thetissue. Fluorometry using Hoechst 33528 (Sigma Aldrich Chemical Company)was used to determine the percent reduction in DNA. The average percentreduction was 99.64%+/−0.07%. In addition, H&E staining and Masson'sTrichrome stain were employed to look for residual nuclei and residualcell remnants, respectively. There were no nuclear remnants seen on theH&E slides and a minimal amount of desirable cytoplasmic proteins. Inaddition immunohistochemistry was performed to demonstrate the absenceof MHCI/II proteins and the presence of alpha smooth muscle actin.

Example 3

Internal mammary artery tissues (two) from an acceptable human donorwere carefully dissected under sterile conditions to remove all visiblefat deposits and side vessels were tied off using nonresorbable suturematerials such that the ties did not occur in close proximity to thelong run of the vessel. Sutures may restrict the devitalization processand the tissues under the sutures were removed following devitalization.For short artery grafts (11 and 8 cm) (FIG. 1), one end of each arterywas cannulated onto the ribbed attachment of the inlet ports and singlesutures used to secure each artery. The arteries were then removed fromthe dissecting solution (RPMI 1640 and papaverine) and one or moreantibiotics and transferred to the processing vessel which had beentemporarily inverted. Prior to closing the processing vessel, a portionof the extracting solution was gently added to the processing vessel andthe inlet port, with attached artery, was then secured. At this point,the processing vessel was turned such that the inlet port was down andthe outlet port was up and the vessel attached to its support rackingsystem via clamps. Sterile disposable tubing was attached to the inletport and to pump tubing in a peristaltic pump. Further, steriledisposable tubing was attached to the inflow side of the peristalticpump and to the solution reservoir, which contained all remainingextracting solution. Total processing solution volume approximated 150ml. Finally, sterile disposable tubing was attached between the top(outlet) port of the processing vessel and the solution reservoir.Sterile, in-line, filters were added at suitable positions in the fluidflow to safeguard sterility during processing. The extracting solutionwas pumped into, through and out of the processing vessel such that flowof fluids through the luminal part of the artery tubule passed into theprocessing vessel to affect constant solution change in the processingvessel and out through the outlet port to a solution reservoir. Byprocessing the artery in an inverted state, air which had been “trapped”in the luminal space of the vein was induced to exit facilitating equalaccess of the processing solutions to the vein tissue being processed.Processing of the artery tissue with the extracting solution wasperformed at 25° C. for 24 hours using a flow rate of the extractingsolution of 100 ml/min. The extracting solution consisted of 50 mMTris-HCl (pH 8.0), 2 mM MgCl₂, 32 mM N-lauroyl sarcosinate, and anendonuclease (BENZONASE®) (325 Units/ml). Following processing withextracting solution, the detergent solution was then replaced withultrapure water that was pumped through the chamber and subsequentlythrough a bed of hydrophobic resin (XAD-4®) and anion exchange resin(AMBERLITE® 910). The processing water solution was circulated (flowrate of 100 mls/min) through the tissue at room temperature (15° C.),for a time period of 36 hours. Following processing with the secondprocessing solution, the artery was removed from the processing vesseland transferred into storage solution of 70% (v:v) pharmaceutical gradeisopropanol in sterile ultrapure water and packaged in a volume of thissolution sufficient to cover the tissue. Fluorometry using Pico green(Molecular Probes) was used to determine the percent reduction in DNA.The average percent reduction was 99.23%+/−0.04%. In addition, H&Estaining and Masson's Trichrome stain were employed to look for residualnuclei and residual cell remnants, respectively. There were no nuclearremnants seen on the H&E slides and a minimal amount of desirablecytoplasmic proteins. In addition immunohistochemistry was performed todemonstrate the absence of MHCI/II proteins and the presence of alphasmooth muscle actin.

Example 4

Aortic and pulmonary tissues (one each) from a heart of an acceptablehuman donor were carefully dissected under sterile conditions to removeall visible fat deposits and cardiac muscle tissue (leaving only a smallbut visible band of cardiac muscle tissue around the proximal end of theconduit). The valves were then removed from the dissecting solution(RPMI 1640) and placed in RPMI 1640 plus 10% fetal calf serum.Subsequently 10% DMSO was added drop-wise to the media, the bag wassealed and cryopreserved in a CryoMed chamber. The tissue was cooled at1° C./min until −40° C. was achieved upon which time the tissue wastransferred to the vapor phase of liquid nitrogen (LN2). At the time ofuse the tissue was removed from the vapor phase LN2, thawed and dilutedusing an AlloFlow® Chamber (AlloFlow is a trademark of LifeNet, VirginiaBeach, Va.) as described in U.S. Pat. Nos. 5,879,876 and 6,326,188,which are incorporated by reference in their entireties herein. Next,the valves were placed in the devitalization chamber, some detergentsolution was added and the chamber sealed. The hydrophobic adsorbentresin and anion exchange resin cartridge was snapped into place. Thevessel was attached to its support racking system via clamps. Steriledisposable tubing was attached to the inlet port and to pump tubing in aperistaltic pump. Further, sterile disposable tubing was attached to theinflow side of the peristaltic pump and to the solution reservoir, whichcontained all remaining extracting solution. Total processing solutionvolume approximated 350 ml. Finally, sterile disposable tubing wasattached between the top (outlet) port of the processing vessel and thesolution reservoir. Sterile, in-line, filters were added at suitablepositions in the fluid flow to safeguard sterility during processing.The extracting solution was pumped into, through and out of theprocessing vessel. Processing of the valve and conduit tissue with theextracting solution was performed at 25° C. for 24 hours using a flowrate of the extracting solution of 250 mls/min. The extracting solutionconsisted of 50 mM Tris-HCl (pH 8.0), 2 mM MgCl₂, 64 mM n-lauroylsarcosinate, and an endonuclease (BENZONASE®) (375 Units/ml). Followingprocessing with the extracting solution, the extracting solution wasreplaced with sterile ultrapure water (350 mls at a pump rate of 250mls/min) being recirculated over a time period of 24 hours and through abed of hydrophobic adsorbent resin (XAD-16) and anion exchange resin(AMBERLITE 410®). Following washing in this final processing step, theheart valves were removed from the processing vessel and transferredinto storage solution of 0.05% chlorine dioxide and 90% glycerol insterile ultrapure water and packaged in a volume of this solutionsufficient to cover the tissue and stored at −80° C. Fluorometry usingPico green (Molecular Probes) was used to determine the percentreduction in DNA. The average percent reduction was 99.13%+/−0.06% forthe conduit, 99.98%+/−0.02% for the leaflets and 99.1%+/−0.05 for themyocardium. In addition, H&E staining and Masson's Trichrome stain wereemployed to look for residual nuclei and residual cell remnants,respectively. There were no nuclear remnants seen on the H&E slides anda minimal amount of desirable cytoplasmic proteins visualized in theconduit and leaflet.

In addition, immunohistochemistry was performed to demonstrate theabsence of MHCI/II proteins in the conduit, leaflet and myocardium andthe presence of alpha smooth muscle actin in the conduit.

Example 5

The devitalized human arterial tissues as produced in Example 3, werestained with Mason Trichrome and histologically examined. Humansaphenous veins were then decellularized using the method as taught inU.S. Pat. No. 4,776,853 (the “'853 patent”), the disclosure of which isincorporated by reference in its entirety herein. Specifically, thefollowing extraction process steps, in accordance with the '853 patent,were employed in this Example:

-   -   1. A variety of arteries were treated: femoral, iliac, carotid,        aortic.    -   2. The vessels were resected and cleaned of adhering connective        tissues and debris prior to extraction.    -   3. Cleaned vessels were immediately placed into the first        extraction solution called Solution A, which consisted of: 10 mM        Tris.HCL and 5 mM EDTA at pH 8.0 supplemented with 50 U/ml        penicillin/streptomycin combination (stock of 10,000 U/ml        penicillin and 10,000 μg/ml streptomycin GIBCO) and 1 μM PMSF        (phenylmethylsulfonyl fluoride—an antiproteolytic agent).        Extraction was carried out with vessels enclosed in Nitex        envelopes in a cylinder at 5° C., with stirring for 24 hours        (range of 24 hours to 48 hours)    -   4. The vessels were placed into the second extraction solution,        Solution B consisting of: 50 mM Tris.HCL, 1.5M KCL, and 1%        Triton X-100 (a non-ionic detergent), 5 mM EDTA at pH 8.0        supplemented with 1 μM PMSF and 50 U/ml penicillin/streptomycin        as in Solution A. Extraction was carried out with vessels in        Nitex envelopes in a cylinder at 5° C. with stirring for 24        hours (range of 24 to 72 hours). The volume ratios of solutions        A or B to tissue were a minimum of 100:1.    -   5. The vessels were washed three times in 100:1 volumes of        either purified (Milli Q system 0.2 u) filtered water or the        same water after autoclaving and then for 30 minutes to 1 hour        in Hanks buffered salt solution (GIBCO) containing 10 mM Hepes        buffer and 50 U/ml penicillin/streptomycin at 37° with rocking.    -   6. The vessels were treated enzymatically as follows: The        vessels were transferred to solutions containing 0.75 mg/15 ml        DNase I (Type III—Sigma) and RNase (Type 1A—Sigma) 1.25 mg/15 ml        and rocked for 4 to 6 hours at 37° C.    -   7. The vessels were washed briefly one time in purified water        for 30 minutes or transferred directly to solution C.    -   8. The vessels were mounted in Nitex envelopes and extracted        with Solution C consisting of 50 mM TRIS.HCl at pH 9.0 with 1%        SDS (sodium dodecyl sulphate) for 24 hours (range 24 to 96        hours) at ambient temperature.    -   9. The vessels were washed in >100:1 volumes of water or saline        at least three times over 24 hours (range 24 to 96 hours).    -   10. The vessels were stored in either Hanks buffered salt        solution with Hepes (10-25 mM) and penicillin and streptomycin        or in phosphate buffered saline with the same antibody at 4° C.        The penicillin and streptomycin concentration was raised to 100        U/ml and 100 μg/ml respectively.

The extraction procedure was initiated by hypotonic lysis of the tissuecells. Antibiotics were included from the onset of the process. No cellpoisons, such as azide were used in this process which was initiated bythe hypotonic lysis of the tissue cells. A high salt, non-ionicdetergent combination was used to extract a substantial proportion ofthe cytoplasmic components. The high salt solution generally included asalt concentration in the range of 1 to 2 Molar of the desired salt. Inaccordance with this Example, the preferred salt was potassium chlorideat 1 to 2 Molar, usually 1.5M. This type of salt will not precipitate incolder solutions at the higher concentrations. It is known from culturedcell work that this combination is gentle and leaves behind a cellcytoskeleton but completely permeabolizes the cell. A combined use ofDNase and RNase was used under physiologic conditions to remove nuclearmaterial, both enzymes being used together to provide an effectiveremoval.

The veins processed according to the '853 patent (the “'853 arteries”)of Example 5 were also stained with Mason Trichrome and histologicallyexamined. Upon examination, the vein sections according to the invention(the “inventive arteries”) stained minimally pink indicating theretention of large molecular weight cytoplasmic proteins in the arterialsoft tissue matrix. On the other hand, the decellularized '853 arteries,did not show any trace of pink staining indicating the absence of largemolecular weight cytoplasmic proteins. Both sets of tissue stainednegatively for the presence of nuclei acids (no black staining) and botharteries stained positively for collagen (green). To confirm theidentity of the pink staining proteins, the inventive arteries and the'853 arteries underwent immunohistochemical staining for alpha smoothmuscle actin and desmin. The inventive arteries of Examples 1-4 stainedweakly for the alpha smooth muscle actin and very weakly for the desminrelative to a cellular positive control. Conversely, the '853 arteriesof Example 5 did not stain positively for either actin or desminconfirming the Massons' Trichrome stain.

Example 6

Pulmonary tissue procured from a sheep was processed according to themethod described in Example 4. At the time of implant, the tissue wasremoved from the −80° C. freezers and placed in an AlloFlow® Chamber tothaw, dilute and rehydrate the graft. The graft was surgically implantedinto the right ventricular outflow tract (RVOT) of a juvenile sheep andremained in vivo for 20 weeks. At explant the valve was examined and wasunremarkable. The valve was sectioned into thirds, each third containinga leaflet, sinus of valsalva and conduit. The H&E staining demonstratedrecellularization of the conduit, sinus of valsalva and one third of theleaflet. Alizarin Red S staining demonstrated all portions of the graftexamined were free from calcification. The TUNEL assay demonstrated allportions of the graft were free from apoptosis. Immunohistochemistry(IHC) for alpha smooth muscle actin demonstrated staining in the mediaof the conduit as expected and somewhat into the media and adventitia asalso expected. IHC for Factor VIII, a specific marker for endothelialcells demonstrated a smooth line of endothelial cells on the basementmembrane of the conduit and on approximately one third of the leaflet.In situ hybridization for Type I collagen demonstrated collagenproduction in the conduit, indicating the recipient cells that hadinfiltrated the matrix and were making autologous collagen and turningover the devitalized donor matrix. The pressure gradients maintained at18 mm Hg throughout the entire implant. These data are is representativeof tissue explained from all 20 sheep implanted.

Example 7

Pulmonary tissue procured from a sheep was processed according to themethod described in Example 4. At the time of implant, the tissue wasremoved from the −80° C. freezers and placed in an AlloFlow® Chamber tothaw, dilute and rehydrate the graft. The graft was fashioned into 2patches and one was surgically implanted into the right ventricularoutflow tract (RVOT) and the other into the descending thoracic aorta(DCA) of a juvenile sheep and remained in vivo for 20 weeks. The patcheswere sewn into a 2 cm defect in the vessel wall. At explant the patcheswere examined and the DCA patches were unremarkable and the RVOT patcheshad adhesions from the lung on the periadventital surface. The patcheswere sectioned into thirds, and each third was further sectioned intofifths. The sections were examined for interactions at the patch andnative tissue junction and patch alone. The H&E staining demonstratedrecellularization of the conduit on all sections examined. Alizarin RedS staining demonstrated all portions of the patch graft examined werefree from calcification. The TUNEL assay demonstrated all portions ofthe patch graft were free from apoptosis. Immunohistochemistry (IHC) foralpha smooth muscle actin demonstrated staining in the media of theconduit as expected and somewhat into the media and adventitia as alsoexpected. IHC for Factor VIII, a specific marker for endothelial cellsdemonstrated a smooth line of endothelial cells on the basement membraneof the conduit. In situ hybridization for Type I collagen demonstratedcollagen production in the conduit, indicating the recipient cells thathad infiltrated the matrix and were making autologous collagen andturning over the devitalized donor matrix.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims. Any references including patents cited herein areincorporated by reference herein in their entirety.

Example 8

Achilles, tibialis and gracilis tendons from an acceptable human donorwere carefully dissected under sterile conditions to remove all bone,fascia and visible fat deposits. The tendons were then removed from thedissecting solution (RPMI 1640) and placed in RPMI 1640 plus 10% fetalcalf serum. Subsequently 10% DMSO was added drop-wise to the media. Thebag was then sealed and cryopreserved in a CryoMed chamber. The tissuewas cooled at 1° C./min until −40° C. was achieved upon which time thetissue was transferred to the vapor phase of liquid nitrogen (LN2). Atthe time of use the tissue was removed from the vapor phase LN2, thawedand diluted using an AlloFlow® Chamber as described in U.S. Pat. Nos.5,879,876 and 6,326,188, which are incorporated by reference in theirentireties herein. Next, the tendons were placed in the devitalizationchamber, some detergent solution was added and the chamber sealed. Thehydrophobic adsorbent resin and anion exchange resin cartridge wassnapped into place. The vessel was attached to its support rackingsystem via clamps. Sterile disposable tubing was attached to the inletport and to pump tubing in a peristaltic pump. Further, steriledisposable tubing was attached to the inflow side of the peristalticpump and to the solution reservoir, which contained all remainingextracting solution. Total processing solution volume approximated 325ml. Finally, sterile disposable tubing was attached between the top(outlet) port of the processing vessel and the solution reservoir.Sterile, in-line, filters were added at suitable positions in the fluidflow to safeguard sterility during processing. The extracting solutionwas pumped into, through and out of the processing vessel. Processing ofthe valve and conduit tissue with the extracting solution was performedat 25° C. for 6 hours using a flow rate of the extracting solution of275 mls/min. The extracting solution consisted of 50 mM Tris-HCl (pH8.0), 2 mM MgCl₂, 64 mM n-lauroyl sarcosinate, and an endonuclease(BENZONASE®) (350 Units/ml). Following processing with the extractingsolution, the extracting solution was replaced with sterile ultrapurewater (325 mls at a pump rate of 275 mls/min) being recirculated over atime period of 18 hours and through a bed of hydrophobic adsorbent resin(XAD-16®) and anion exchange resin (AMBERLITE 410®). Following washingin this final processing step, the tendons were removed from theprocessing vessel and transferred into storage solution of 0.05%chlorine dioxide and 90% glycerol in sterile ultrapure water andpackaged in a volume of this solution sufficient to cover the tissue andstored at −80° C. Fluorometry using Pico green (Molecular Probes) wasused to determine the percent reduction in DNA. The average percentreduction was 99.89%+/−0.2%. In addition, H&E staining and Masson'sTrichrome stain were employed to look for residual nuclei and residualcell remnants, respectively. There were no nuclear remnants seen on theH&E slides, small amounts of cell membranes and a minimal amount ofdesirable cytoplasmic proteins visualized in the tendon.

Example 9

Gracilis tendon tissue procured from a sheep was processed according tothe method described in Example 8. At the time of implant, the tissuewas removed from the −80° C. freezers and placed in an AlloFlow® Chamberto thaw, dilute and rehydrate the graft. The graft was surgicallyimplanted as anterior cruciate ligament repair in adult female sheep andremained in viva for 20 weeks. The surgery was a femoral tunnel withcross pin fixation for 10 animals and a tibial tunnel with staplefixation for 10 animals. At explant the tendon was examined and wasunremarkable with respect to inflammatory changes such as fibrosis;however, the origin and insertion had calcified and incorporated intothe patella. The tendon was sectioned into thirds, to examine theorigin, insertion and middle of tendon. The H&E staining demonstratedrecellularization of the tendon. The area of origin and insertionportions of the tendon also demonstrated osteoblasts, and areas oflamellae with osteocytes in the lacunae. Alizarin Red S stainingdemonstrated calcification localized to the insertion and origin pinsites within the tendons. The TUNEL assay demonstrated all portions ofthe graft were free from apoptosis. In situ hybridization for Type Icollagen demonstrated collagen production in the tendon indicating therecipient cells that had infiltrated the tendon were making autologouscollagen and turning over the devitalized donor matrix. These data arerepresentative of tissue explanted from all 20 sheep implanted.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims. Any references including patents cited herein areincorporated by reference herein in their entirety.

What is claimed:
 1. A process for preparing a devitalized soft tissuegraft for implantation into a mammalian system from a soft tissue samplecomprising large molecular weight cytoplasmic proteins, the processcomprising subjecting the soft tissue sample to an extracting solutioncomprising a non-denaturing detergent in an amount effective fordevitalizing the soft tissue sample without removing the large molecularweight cytoplasmic proteins from the soft tissue sample, wherein thenon-denaturing detergent is n-lauroyl sarcosinate, whereby a devitalizedsoft tissue graft is obtained, and wherein the devitalized soft tissuegraft retains the large molecular weight cytoplasmic proteins.
 2. Theprocess of claim 1, wherein the extracting solution further comprises anendonuclease.
 3. The process of claim 2, wherein the endonuclease is arecombinant endonuclease.
 4. The process of claim 1, wherein theextracting solution further comprises a decontaminating agent.
 5. Theprocess of claim 1, further comprising processing the devitalized softtissue graft with ion exchange resins to remove the detergent.
 6. Theprocess of claim 1, further comprising washing the devitalized softtissue graft.
 7. The process of claim 1, wherein the process does notinclude deoxycholic acid or cholic acid.
 8. The process of claim 1,wherein the devitalized soft tissue graft is for a cardiovascularapplication.
 9. The process of claim 1, wherein the devitalized softtissue graft is for a musculoskeletal application.
 10. The process ofclaim 1, wherein the large molecular weight cytoplasmic proteins isselected from the group consisting of actin, desmin and vimentin.